Biomass compositions for increasing sweetness of fruit and methods therefor

ABSTRACT

Biomass compositions and methods for increasing sweetness of fruit of a fruiting plant by administering to the fruiting plant, seedling, or seed, a liquid composition treatment comprising a culture of microalgae cells are disclosed. The liquid composition may comprise pasteurized  Chlorella  cells only,  Aurantiochytrium acetophilum  HS399 cells only, or a combination of  Chlorella  and  Aurantiochytrium acetophilum  HS399 cells that are pasteurized at a temperature between 65° C.-90° C. The administration may comprise contacting soil in the immediate vicinity of the fruiting plant, seedling, or seed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of Provisional Application No. 62/584,311 entitled “BIOMASS COMPOSITIONS,” which was filed on Nov. 10, 2017 in the name of the Applicant and which is incorporated herein in full by reference. This Application also claims the benefit of Provisional Application No. 62/680,373 entitled “BIOMASS COMPOSITIONS,” which was filed on Jun. 4, 2018 in the name of the Applicant and which is incorporated herein in full by reference.

FIELD OF THE INVENTION

The present invention generally relates to agriculture and, more specifically, to biomass compositions and methods for increasing sweetness in fruits of fruiting plants.

BACKGROUND OF THE INVENTION

The growth of a plant is a complex physiological process involving inputs and pathways in the roots, shoots, and leaves. Whether at a commercial or home garden scale, growers are constantly striving to optimize the yield and quality of plants, which may include the sweetness of fruits from fruiting plants.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key factors or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

Embodiments of the invention relate to a composition for enhancing at least one plant characteristic. The composition can include a microalgae biomass that includes at least one species of microalgae. The composition can include a microalgae biomass that includes at least two species of microalgae. The composition can cause synergistic enhancement of at least one plant characteristic.

In some embodiments, the microalgae species can include Chlorella, Schizochytrium, Thraustochytrium, Oblongichytrium and/or Aurantiochytrium acetophilum HS399. In other embodiments, the microalgae species can include Botryococcus, Chlamydomonas, Scenedesmus, Pavlova, Phaeodactylum, Nannochloropsis, Spirlulina, Galdieria, Haematococcus, Isochrysis, Porphyridium, Tetraselmis, and/or the like.

In some embodiments, the microalgae biomass can include whole biomass and/or residual biomass. Whole biomass includes substantially all components and fractions of the cells from which the whole biomass is derived. Residual or extracted biomass can be any remaining biomass after extraction and/or removal of one or more components of a whole biomass.

In some embodiments, the composition can include one species of microalgae. In some embodiments, the composition can include a first species of microalgae and a second species of microalgae. The ratio of the first species of microalgae and the second species of microalgae can be between about 25:75, 50:50, or 75:25.

In some embodiments, the first species of microalgae may be Chlorella and the second species of microalgae may be Aurantiochytrium acetophilum HS399. In some embodiments, the ratio of Chlorella and Aurantiochytrium acetophilum HS399 may range between about 25:75 to 75:25. For example, the ratio of Chlorella and Aurantiochytrium acetophilum HS399 may be about 25:75, 50:50, or 75:25. In some embodiments, the Chlorella is whole biomass and Aurantiochytrium acetophilum HS399 is residual/extracted biomass. In some embodiments, the Aurantiochytrium acetophilum HS399 is whole biomass and Chlorella is residual/extracted biomass. In some embodiments, the Chlorella and Aurantiochytrium acetophilum HS399 are both whole biomass and in other embodiments the Chlorella and Aurantiochytrium acetophilum HS399 are both residual/extracted biomass.

Some embodiments of the invention relate to a method of plant enhancement comprising administering to a plant, seedling, seed, or soil the composition treatment, wherein the composition treatment enhances at least one plant characteristic. In some embodiments, the composition is applied when the plant is under salt stress conditions, temperature stress conditions, and/or the like.

Embodiments of the invention relate to a method of plant enhancement comprising administering a composition treatment comprising at least one microalgae species to soil. The administering can be by soil drench at the time of seeding. The method can include growing the plant to a transplant stage. The method can include transferring the plant at the transplant stage from an initial container to a larger container or a field, or the like. In some embodiments the plant at the transplant stage has at least one enhanced plant characteristic. The enhanced plant characteristic can be improved root density, improved root area, enhanced plant vigor, enhanced plant growth rate, enhanced plant maturation, and/or enhanced shoot development. After the transfer, the plant may have at least one enhanced plant characteristic. The composition treatment can include at least one microalgae species such as Botryococcus, Chlorella, Chlamydomonas, Scenedesmus, Pavlova, Phaeodactylum, Nannochloropsis, Aurantiochytrium, Spirlulina, Galdieria, Haematococcus, Isochrysis, Porphyridium, Schizochytrium, Tetraselmis, and/or the like.

In some of the embodiments and Examples below, the microalgae composition may be applied to the soil of the fruiting plant by drenching the soil initially at the time of transplant and then subsequently every two weeks (once every 14 days) after transplant until harvest.

In one embodiment of the present invention, a method of increasing sweetness of fruit of a fruiting plant is disclosed. The method comprises the step of administering to the fruiting plant a liquid composition treatment comprising a culture of microalgae, the microalgae comprising at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells in an effective amount to increase total dissolved sugars in the fruit of a population of such fruiting plants compared to a substantially identical population of untreated fruiting plants.

In another embodiment of the present invention, a method of increasing sweetness of fruit of a fruiting plant is disclosed. The method comprises the step of administering to the fruiting plant, seedling, seed a liquid composition treatment comprising a culture of microalgae, the microalgae comprising at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells in an effective amount to increase total dissolved sugars in the fruit of a population of such fruiting plants by 2-32% compared to fruit of a substantially identical population of untreated fruiting plants, wherein administering comprises contacting soil in the immediate vicinity of the fruiting plant, seedling, or seed with an effective amount of the liquid composition treatment by drip irrigation.

In another embodiment of the present invention, a method of increasing sweetness of fruit of a fruiting plant is disclosed. The method comprises the steps of: providing a liquid composition treatment comprising a culture of microalgae, the microalgae comprising at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells; diluting the liquid composition treatment to contain between 0.95 g-15 g per gallon of the at least one of pasteurized Chlorella cells and Aurantiochytrium acetophilum HS399 cells; and administering the liquid composition treatment to a fruiting plant, seedling, or seed, in an effective amount to increase total dissolved sugars in the fruit of a population of such fruiting plants compared to a substantially identical population of untreated fruiting plants.

To the accomplishment of the foregoing and related ends, the following description and annexed drawings set forth certain illustrative aspects and implementations. These are indicative of but a few of the various ways in which one or more aspects may be employed. Other aspects, advantages and novel features of the disclosure will become apparent from the following detailed description when considered in conjunction with the annexed drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present application is further detailed with respect to the following drawings. These figures are not intended to limit the scope of the present application, but rather, illustrate certain attributes thereof.

FIG. 1 is a graph showing a comparison of the effects of several microalgae compositions on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 2 is a table showing a comparison of the effects of several microalgae compositions on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 3 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 2 on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 4 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 2 on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 5 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 2 on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 6 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 2 on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 7 is a graph showing a comparison of the effects of several microalgae compositions on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 8 is a graph showing a comparison of the effects of several microalgae compositions on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 9 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 8 on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 10 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 8 on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product.

FIG. 11 is a graph showing a comparison of the effects of several microalgae compositions on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 12 is a graph showing a comparison of the effects of several microalgae compositions on strawberry quality, wherein the effects are observed in an increase in strawberry sweetness (% brix) relative to the UTC and a seaweed commercial reference product;

FIG. 13 is a graph showing the effects of a microalgae composition on mature green bell pepper quality, wherein the effects are observed in an increase in bell pepper sweetness (% brix) relative to the UTC;

FIG. 14 is a graph showing the effects of the microalgae composition of FIG. 13 on mature green bell pepper quality, wherein the effects are observed in an increase in bell pepper sweetness (% brix) relative to the UTC;

FIG. 15 is a graph showing the effects of the microalgae composition of FIG. 13 on mature green bell pepper quality, wherein the effects are observed in an increase in bell pepper sweetness (% brix) relative to the UTC;

FIG. 16 is a graph showing the effects of the microalgae composition of FIG. 13 on red ripe bell pepper quality, wherein the effects are observed in an increase in bell pepper sweetness (% brix) relative to the UTC;

FIG. 17 is a graph showing the effects of the microalgae composition of FIG. 13 on red ripe bell pepper quality, wherein the effects are observed in an increase in bell pepper sweetness (% brix) relative to the UTC; and

FIG. 18 is a graph showing the effects of the microalgae composition of FIG. 13 on red ripe bell pepper quality, wherein the effects are observed in an increase in bell pepper sweetness (% brix) relative to the UTC.

DETAILED DESCRIPTION OF THE INVENTION

The description set forth below in connection with the appended drawings is intended as a description of presently preferred embodiments of the disclosure and is not intended to represent the only forms in which the present disclosure may be constructed and/or utilized. The description sets forth the functions and the sequence of steps for constructing and operating the disclosure in connection with the illustrated embodiments. It is to be understood, however, that the same or equivalent functions and sequences may be accomplished by different embodiments that are also intended to be encompassed within the spirit and scope of this disclosure.

Many plants can benefit from the application of liquid compositions that provide a bio-stimulatory effect. Non-limiting examples of plant families that can benefit from such compositions include plants from the following: Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae, Vitaceae, Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae, Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae, Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae (Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae, Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae, Rubiaceae, Papveraceae, Illiciaceae Grossulariaceae, Myrtaceae, Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae), Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae, Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae, Piperaceae, Proteaceae, and Cannabaceae.

The Solanaceae plant family includes a large number of agricultural crops, medicinal plants, spices, and ornamentals in its over 2,500 species. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Manoliopsida (class), Asteridae (subclass), and Solanales (order), the Solanaceae family includes, but is not limited to, potatoes, tomatoes, eggplants, various peppers, tobacco, and petunias. Plants in the Solanaceae can be found on all the continents, excluding Antarctica, and thus have a widespread importance in agriculture across the globe.

The Rosaceae plant family includes flowering plants, herbs, shrubs, and trees. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Magnoliopsida (class), Rosidae (subclass), and Rosales (order), the Rosaceae family includes, but is not limited to, almond, apple, apricot, blackberry, cherry, nectarine, peach, plum, raspberry, strawberry, and quince.

The Fabaceae plant family (also known as the Leguminosae) comprises the third largest plant family with over 18,000 species, including a number of important agricultural and food plants. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Manoliopsida (class), Rosidae (subclass), and Fabales (order), the Fabaceae family includes, but is not limited to, soybeans, beans, green beans, peas, chickpeas, alfalfa, peanuts, sweet peas, carob, and liquorice. Plants in the Fabaceae family can range in size and type, including but not limited to, trees, small annual herbs, shrubs, and vines, and typically develop legumes. Plants in the Fabaceae family can be found on all the continents, excluding Antarctica, and thus have a widespread importance in agriculture across the globe. Besides food, plants in the Fabaceae family can be used to produce natural gums, dyes, and ornamentals.

The Poaceae plant family supplies food, building materials, and feedstock for fuel processing. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Liliopsida (class), Commelinidae (subclass), and Cyperales (order), the Poaceae family includes, but is not limited to, flowering plants, grasses, and cereal crops such as barely, corn, lemongrass, millet, oat, rye, rice, wheat, sugarcane, and sorghum. Types of turf grass found in Arizona include, but are not limited to, hybrid Bermuda grasses (e.g., 328 tifgrn, 419 tifway, tif sport).

The Vitaceae plant family includes flowering plants and vines. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Magnoliopsida (class), Rosidae (subclass), and Rhammales (order), the Vitaceae family includes, but is not limited to, grapes.

Particularly important in the production of fruit from plants is the beginning stage of growth where the plant emerges and matures into establishment. A method of treating a seed, seedling, or plant to directly improve the germination, emergence, and maturation of the plant; or to indirectly enhance the microbial soil community surrounding the seed or seedling is therefore valuable starting the plant on the path to marketable production. The standard typically used for assessing emergence is the achievement of the hypocotyl stage, where a stem is visibly protruding from the soil. The standard typically used for assessing maturation is the achievement of the cotyledon stage, where two leaves visibly form on the emerged stem. Some botanists view the beginning of maturation as starting at when the first true leaf emerges beyond the cotyledon stage, as the cotyledons are already pre-formed in the seed prior to germination. Some botanists see maturation as a long phase that proceeds until full reproductive potential has been achieved.

Important in the production of fruit from plants is the yield and quality of fruit, which can be quantified as the number, weight, color, firmness, ripeness, sweetness, moisture, degree of insect infestation, degree of disease or rot, degree of sunburn of the fruit. A method of treating a plant to directly improve the characteristics of the plant, or to indirectly enhance the chlorophyll level of the plant for photosynthetic capabilities and health of the plant's leaves, roots, and shoot to enable robust production of fruit is therefore valuable in increasing the efficiency of marketable production. Marketable and unmarketable designations can apply to both the plant and fruit, and can be defined differently based on the end use of the product, such as but not limited to, fresh market produce and processing for inclusion as an ingredient in a composition. The marketable determination can assess such qualities as, but not limited to, color, insect damage, blossom end rot, softness, and sunburn. The term “total production” can incorporate both marketable and unmarketable plants and fruit. The ratio of marketable plants or fruit to unmarketable plants or fruit can be referred to as “utilization” and expressed as a percentage. The utilization can be used as an indicator of the efficiency of the agricultural process as it shows the successful production of marketable plants or fruit, which will be obtain the highest financial return for the grower, whereas total production will not provide such an indication.

To achieve such improvements in emergence, maturation, and yield of plants, a method to treat such seeds and plants, and soil with a low-concentration microalgae-based composition, in a dried or liquid solution form was developed. Microalgae can be grown in heterotrophic, mixotrophic, and phototrophic conditions. Culturing microalgae in heterotrophic conditions comprises supplying organic carbon (e.g., acetic acid, acetate, glucose) to cells in an aqueous culture medium comprising trace metals and nutrients (e.g., nitrogen, phosphorus). Culturing microalgae in mixotrophic conditions comprises supplying light and organic carbon (e.g., acetic acid, acetate, glucose) to cells in an aqueous culture medium comprising trace metals and nutrients (e.g., nitrogen, phosphorus). Culturing microalgae in phototrophic conditions comprises supplying light and inorganic carbon (e.g., carbon dioxide) to cells in an aqueous culture medium comprising trace metals and nutrients (e.g., nitrogen, phosphorus).

In some embodiments, the microalgae cells can be harvested from a culture and used as whole cells in a liquid composition for application to seeds and plants, while in other embodiments the harvested microalgae cells can be subjected to downstream processing and the resulting biomass or extract can be used in a dried composition (e.g., powder, pellet) or a liquid composition (e.g., suspension, solution) for application to plants, soil, or a combination thereof. Non-limiting examples of downstream processing comprise: drying the cells, lysing the cells, and subjecting the harvested cells to a solvent or supercritical carbon dioxide extraction process to isolate an oil or protein. In some embodiments, the extracted (i.e., residual) biomass remaining from an extraction process can be used alone or in combination with other microalgae or extracts in a liquid composition for application to plants, soil, or a combination thereof. By subjecting the microalgae to an extraction process the resulting biomass is transformed from a natural whole state to a lysed condition where the cell is missing a significant amount of the natural components, thus differentiating the extracted microalgae biomass from that which is found in nature. Excreted products from the microalgae can also be isolated from a microalgae culture using downstream processing methods.

In some embodiments, microalgae can be the predominant active ingredient source in the composition. In some embodiments, the microalgae population of the composition can include whole biomass, substantially extracted biomass, excreted products (e.g., EPS), extracted protein, or extracted oil. In some embodiments, microalgae include at least 99% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 95% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 90% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 80% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 70% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 60% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 50% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 40% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 30% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 20% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 10% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 5% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 1% of the active ingredient sources of the composition. In some embodiments, the composition lacks any detectable amount of any other active ingredient source other than microalgae.

In some embodiments, microalgae biomass, excreted products, or extracts can also be mixed with biomass or extracts from other plants, microalgae, macroalgae, seaweeds, and kelp. In some embodiments, microalgae biomass, excreted products, or extracts can also be mixed with fish oil. Non-limiting examples of other plants, macroalgae, seaweeds, and kelp fractions that can be combined with microalgae cells can include species of Lemna, Gracilaria, Kappaphycus, Ascophyllum, Macrocystis, Fucus, Laminaria, Sargassum, Turbinaria, and Durvilea. In further embodiments, the extracts can comprise, but are not limited to, liquid extract from a species of Kappaphycus. In some embodiments, the extracts can include 50% or less by volume of the composition. In some embodiments, the extracts can include 40% or less by volume of the composition. In some embodiments, the extracts can include 30% or less by volume of the composition. In some embodiments, the extracts can include 20% or less by volume of the composition. In some embodiments, the extracts can include 10% or less by volume of the composition. In some embodiments, the extracts can include 5% or less by volume of the composition. In some embodiments, the extracts can include 4% or less by volume of the composition. In some embodiments, the extracts can include 3% or less by volume of the composition. In some embodiments, the extracts can include 2% or less by volume of the composition. In some embodiments, the extracts can include I % or less by volume of the composition.

The term “microalgae” refers to microscopic single cell organisms such as microalgae, cyanobacteria, algae, diatoms, dinoflagellates, freshwater organisms, marine organisms, or other similar single cell organisms capable of growth in phototrophic, mixotrophic, or heterotrophic culture conditions.

In some embodiments, microalgae biomass, excreted product, or extracts can also be sourced from multiple types of microalgae, to make a composition that is beneficial when applied to plants or soil. Non-limiting examples of microalgae that can be used in the compositions and methods of the present invention include microalgae in the classes: Eustigmatophyceae, Chlorophyceae, Prasinophyceae, Haptophyceae, Cyanidiophyceae, Prymnesiophyceae, Porphyridiophyceae, Labyrinthulomycetes, Trebouxiophyceae, Bacillariophyceae, and Cyanophyceae. The class Cyanidiophyceae includes species of Galdieria. The class Chlorophyceae includes species of Haematococcus, Scenedesmus, Chlamydomonas, and Micractinium. The class Prymnesiophyceae includes species of Isochrysis and Pavlova. The class Eustigmatophyceae includes species of Nannochloropsis. The class Porphyridiophyceae includes species of Porphyridium. The class Labyrinthulomycetes includes species of Schizochytrium and Aurantiochytrium. The class Prasinophyceae includes species of Tetraselmis. The class Trebouxiophyceae includes species of Chlorella and Botryococcus. The class Bacillariophyceae includes species of Phaeodactylum. The class Cyanophyceae includes species of Spirulina.

Non-limiting examples of microalgae genus and species that can be used in the compositions and methods of the present invention include: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Aurantiochytrium sp., Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomonas sp., Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolate, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Galdieria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis a.ff galbana, Isochrysis galbana, Lepocinclis, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis camerae, Pleurochrysis dentate, Pleurochrysis sp., Porphyridium sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

Analysis of the DNA sequence of the strain of Chlorella sp. described in the specification was done in the NCBI 18s rDNA reference database at the Culture Collection of Algae at the University of Cologne (CCAC) showed substantial similarity (i.e., greater than 95%) with multiple known strains of Chlorella and Micractinium. Those of skill in the art will recognize that Chlorella and Micractinium appear closely related in many taxonomic classification trees for microalgae, and strains and species may be re-classified from time to time. Thus, for references throughout the instant specification for Chlorella sp., it is recognized that microalgae strains in related taxonomic classifications with similar characteristics to the reference Chlorella sp. strain would reasonably be expected to produce similar results.

Additionally, taxonomic classification has also been in flux for organisms in the genus Schizochytrium. Some organisms previously classified as Schizochytrium have been reclassified as Aurantiochytrium, Thraustochytrium, or Oblongichytrium. See Yokoyama et al. Taxonomic rearrangement of the genus Schizochytrium [sensu lato] based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thrausochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience (2007) 48:199-211. Those of skill in the art will recognize that Schizochytrium, Aurantiochytrium, Thraustochytrium, and Oblongichytrium appear closely related in many taxonomic classification trees for microalgae, and strains and species may be re-classified from time to time. Thus, for references throughout the instant specification for Schizochytrium, it is recognized that microalgae strains in related taxonomic classifications with similar characteristics to Schizochytrium would reasonably be expected to produce similar results. Furthermore, with respect to all references to “Aurantiochytrium acetophilum HS399” herein, pursuant to the requirements of the Budapest Treaty, a live culture of the Aurantiochytrium acetophilum HS399 microalgae strain described herein was deposited on Sep. 12, 2019 at National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA and received accession number 201909001.

By artificially controlling aspects of the microalgae culturing process such as the organic carbon feed (e.g., acetic acid, acetate), oxygen levels, pH, and light, the culturing process differs from the culturing process that microalgae experiences in nature. In addition to controlling various aspects of the culturing process, intervention by human operators or automated systems occurs during the non-axenic mixotrophic culturing of microalgae through contamination control methods to prevent the microalgae from being overrun and outcompeted by contaminating organisms (e.g., fungi, bacteria). Contamination control methods for microalgae cultures are known in the art and such suitable contamination control methods for non-axenic mixotrophic microalgae cultures are disclosed in W02014/074769A2 (Ganuza, et al.), hereby incorporated by reference. By intervening in the microalgae culturing process, the impact of the contaminating microorganisms can be mitigated by suppressing the proliferation of containing organism populations and the effect on the microalgal cells (e.g., lysing, infection, death, clumping). Thus, through artificial control of aspects of the culturing process and intervening in the culturing process with contamination control methods, the microalgae culture produced as a whole and used in the described inventive compositions differs from the culture that results from a microalgae culturing process that occurs in nature.

During the mixotrophic culturing process the microalgae culture can also include cell debris and compounds excreted from the microalgae cells into the culture medium. The output of the microalgae mixotrophic culturing process provides the active ingredient for composition that is applied to plants for improving yield and quality without separate addition to or supplementation of the composition with other active ingredients not found in the mixotrophic microalgae whole cells and accompanying culture medium from the mixotrophic culturing process such as, but not limited to: microalgae extracts, macroalgae, macroalgae extracts, liquid fertilizers, granular fertilizers, mineral complexes (e.g., calcium, sodium, zinc, manganese, cobalt, silicon), fungi, bacteria, nematodes, protozoa, digestate solids, chemicals (e.g., ethanolamine, borax, boric acid), humic acid, nitrogen and nitrogen derivatives, phosphorus rock, pesticides, herbicides, insecticides, enzymes, plant fiber (e.g., coconut fiber).

In some embodiments, the microalgae can be previously frozen and thawed before inclusion in the liquid composition. In some embodiments, the microalgae may not have been subjected to a previous freezing or thawing process. In some embodiments, the microalgae whole cells have not been subjected to a drying process. The cell walls of the microalgae of the composition have not been lysed or disrupted, and the microalgae cells have not been subjected to an extraction process or process that pulverizes the cells. The microalgae whole cells are not subjected to a purification process for isolating the microalgae whole cells from the accompanying constituents of the culturing process (e.g., trace nutrients, residual organic carbon, bacteria, cell debris, cell excretions), and thus the whole output from the microalgae culturing process comprising whole microalgae cells, culture medium, cell excretions, cell debris, bacteria, residual organic carbon, and trace nutrients, is used in the liquid composition for application to plants. In some embodiments, the microalgae whole cells and the accompanying constituents of the culturing process are concentrated in the composition. In some embodiments, the microalgae whole cells and the accompanying constituents of the culturing process are diluted in the composition to a low concentration. The microalgae whole cells of the composition are not fossilized. In some embodiments, the microalgae whole cells are not maintained in a viable state in the composition for continued growth after the method of using the composition in a soil or foliar application. In some embodiments, the microalgae base composition can be biologically inactive after the composition is prepared. In some embodiments, the microalgae base composition can be substantially biologically inactive after the composition is prepared. In some embodiments, the microalgae base composition can increase in biological activity after the prepared composition is exposed to air.

In some embodiments, a liquid composition can include low concentrations of bacteria contributing to the solids percentage of the composition in addition to the microalgae cells. Examples of bacteria found in non-axenic mixotrophic conditions can be found in W02014/074769A2 (Ganuza, et al.), hereby incorporated by reference. A live bacteria count can be determined using methods known in the art such as plate counts, plates counts using Petrifilm available from 3M (St. Paul, Minn.), spectrophotometric (turbidimetric) measurements, visual comparison of turbidity with a known standard, direct cell counts under a microscope, cell mass determination, and measurement of cellular activity. Live bacteria counts in a non-axenic mixotrophic microalgae culture can range from I0⁴ to I0⁹ CFU/mL, and can depend on contamination control measures taken during the culturing of the microalgae. The level of bacteria in the composition can be determined by an aerobic plate count which quantifies aerobic colony forming units (CFU) in a designated volume. In some embodiments, the composition includes an aerobic plate count of 40,000-400,000 CFU/mL. In some embodiments, the composition includes an aerobic plate count of 40,000-100,000 CFU/mL. In some embodiments, the composition includes an aerobic plate count of 100,000-200,000 CFU/mL. In some embodiments, the composition includes an aerobic plate count of 200,000-300,000 CFU/mL. In some embodiments, the composition includes an aerobic plate count of 300,000-400,000 CFU/mL.

In some embodiments, the microalgae based composition can be supplemented with a supplemental nutrient such as nitrogen, phosphorus, or potassium to increase the levels within the composition to at least 1% of the total composition (i.e., addition of N, P, or K to increase levels at least 1-0-0, 0-1-0, 0-0-1, or combinations thereof). In some embodiments, the microalgae composition can be supplemented with nutrients such as, but not limited to, calcium, magnesium, silicon, sulfur, iron, manganese, zinc, copper, boron, molybdenum, chlorine, sodium, aluminum, vanadium, nickel, cerium, dysprosium, erbium, europium, gadolinium, holmium, lanthanum, lutetium, neodymium, praseodymium, promethium, samarium, scandium, terbium, thulium, ytterbium, and yttrium. In some embodiments, the supplemented nutrient is not uptaken, chelated, or absorbed by the microalgae. In some embodiments, the concentration of the supplemental nutrient can include 1-50 g per 100 g of the composition.

A liquid composition comprising microalgae can be stabilized by heating and cooling in a pasteurization process. As shown in the Examples, the inventors found that the active ingredients of the microalgae based composition maintained effectiveness in at least one characteristic of a plant after being subjected to the heating and cooling of a pasteurization process. In other embodiments, liquid compositions with whole cells or processed cells (e.g., dried, lysed, extracted) of microalgae cells may not need to be stabilized by pasteurization. For example, microalgae cells that have been processed, such as by drying, lysing, and extraction, or extracts can include such low levels of bacteria that a liquid composition can remain stable without being subjected to the heating and cooling of a pasteurization process.

In some embodiments, the composition can be heated to a temperature in the range of 50-130° C. In some embodiments, the composition can be heated to a temperature in the range of 55-65° C. In some embodiments, the composition can be heated to a temperature in the range of 58-62° C. In some embodiments, the composition can be heated to a temperature in the range of 50-60° C. In some embodiments, the composition can be heated to a temperature in the range of 60-90° C. In some embodiments, the composition can be heated to a temperature in the range of 70-80° C. In some embodiments, the composition can be heated to a temperature in the range of 100-150° C. In some embodiments, the composition can be heated to a temperature in the range of 120-130° C.

In some embodiments, the composition can be heated for a time period in the range of 1-150 minutes. In some embodiments, the composition can be heated for a time period in the range of 110-130 minutes. In some embodiments, the composition can be heated for a time period in the range of 90-100 minutes. In some embodiments, the composition can be heated for a time period in the range of 100-110 minutes. In some embodiments, the composition can be heated for a time period in the range of 110-120 minutes. In some embodiments, the composition can be heated for a time period in the range of 120-130 minutes. In some embodiments, the composition can be heated for a time period in the range of 130-140 minutes. In some embodiments, the composition can be heated for a time period in the range of 140-150 minutes. In some embodiments, the composition is heated for less than 15 min. In some embodiments, the composition is heated for less than 2 min.

After the step of heating or subjecting the liquid composition to high temperatures is complete, the compositions can be cooled at any rate to a temperature that is safe to work with. In one non-limiting embodiment, the composition can be cooled to a temperature in the range of 35-45° C. In some embodiments, the composition can be cooled to a temperature in the range of 36-44° C. In some embodiments, the composition can be cooled to a temperature in the range of 37-43° C. In some embodiments, the composition can be cooled to a temperature in the range of 38-42° C. In some embodiments, the composition can be cooled to a temperature in the range of 39-41° C. In further embodiments, the pasteurization process can be part of a continuous production process that also involves packaging, and thus the liquid composition can be packaged (e.g., bottled) directly after the heating or high temperature stage without a cooling step.

In some embodiments, the composition can include 5-30% solids by weight of microalgae cells (i.e., 5-30 g of microalgae cells/100 mL of the liquid composition). In some embodiments, the composition can include 5-20% solids by weight of microalgae cells. In some embodiments, the composition can include 5-15% solids by weight of microalgae cells. In some embodiments, the composition can include 5-10% solids by weight of microalgae cells. In some embodiments, the composition can include 10-20% solids by weight of microalgae cells. In some embodiments, the composition can include 10-20% solids by weight of microalgae cells. In some embodiments, the composition can include 20-30% solids by weight of microalgae cells. In some embodiments, further dilution of the microalgae cells percent solids by weight can occur before application for low concentration applications of the composition.

In some embodiments, the composition can include less than 1% by weight of microalgae biomass or extracts (i.e., less than 1 g of microalgae derived product/100 mL of the liquid composition). In some embodiments, the composition can include less than 0.9% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.8% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.7% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.6% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.5% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.4% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.3% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.2% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.0001% by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.001% by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.01% by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.0001-1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.0001-0.001% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.001-0.01% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.01-0.1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.1-1% by weight of microalgae biomass or extracts.

In some embodiments, an application concentration of 0.1% of microalgae biomass or extract equates to 0.04 g of microalgae biomass or extract in 40 mL of a composition. While the desired application concentration to a plant can be 0.1% of microalgae biomass or extract, the composition can be packaged as a 10% concentration (0.4 mL in 40 mL of a composition). Thus, a desired application concentration of 0.1% would require 6,000 mL of the 10% microalgae biomass or extract in the 100 gallons of water applied to the assumption of 15,000 plants in an acre, which is equivalent to an application rate of about 1.585 gallons per acre. In some embodiments, a desired application concentration of 0.01% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.159 gallons per acre. In some embodiments, a desired application concentration of 0.001% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.016 gallons per acre. In some embodiments, a desired application concentration of 0.0001% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.002 gallons per acre.

In another non-limiting embodiment, correlating the application of the microalgae biomass or extract on a per plant basis using the assumption of 15,000 plants per acre, the composition application rate of 1 gallon per acre is equal to about 0.25 mL per plant=0.025 g per plant=25 mg of microalgae biomass or extract per plant. The water requirement assumption of 100 gallons per acre is equal to about 35 mL of water per plant. Therefore, 0.025 g of microalgae biomass or extract in 35 mL of water is equal to about 0.071 g of microalgae biomass or extract per 100 mL of composition equates to about a 0.07% application concentration. In some embodiments, the microalgae biomass or extract based composition can be applied at a rate in a range as low as about 0.001-10 gallons per acre, or as high as up to 150 gallons per acre.

In some of the embodiments and Examples below, the applications were performed using a 10% solids solution by weight microalgae composition. For greenhouse trials, the rates vary and essentially refer to how much volume of the 10% solids solution was added in a given volume of water (e.g. 9 ml/gal-150 ml/gal). For field trials, the rates are indicated in gal/acre and the amount of carrier water would be determined according to user preference. For field trials, the application rate may range between 0.25 gal/acre-2 gal/acre. For example, in the greenhouse trial where the application rate is 9 ml/gal, the microalgae composition would contain 0.95 g of microalgae/gal and where the application rate is 150 ml/gal, the microalgae composition would contain 15 g of microalgae/gal. In the field trials, where the application rate of the microalgae composition is 0.25 gal/acre, the equivalent expressed in total grams of solid microalgae would be 100 g microalgae/acre; where the application rate of the microalgae composition is 0.5 gal/acre, the equivalent expressed in total grams of solid microalgae would be 200 g microalgae/acre; where the application rate of the microalgae composition is 1.0 gal/acre, the equivalent expressed in total grams of solid microalgae would be 400 g microalgae/acre; and where the application rate of the microalgae composition is 2.0 gal/acre, the equivalent expressed in total grams of solid microalgae would be 800 g microalgae/acre.

Overall, as shown in the embodiments and Examples below, the microalgae composition may comprise between 0.95 g-15 g of microalgae per gallon, as it is common practice for growers to use between 100-250 gallons of liquid carrier volume/acre. It should be clearly understood, however, that modifications to the amount of microalgae per gallon may be adjusted upwardly or downwardly to compensate for greater than 250 gallons of liquid carrier volume/acre or less than 100 gallons of liquid carrier volume/acre.

In some embodiments, stabilizing means that are not active regarding the improvement of plant germination, emergence, maturation, quality, and yield, but instead aid in stabilizing the composition can be added to prevent the proliferation of unwanted microorganisms (e.g., yeast, mold) and prolong shelf life. Such inactive but stabilizing means can include an acid, such as but not limited to phosphoric acid or citric acid, and a yeast and mold inhibitor, such as but not limited to potassium sorbate. In some embodiments, the stabilizing means are suitable for plants and do not inhibit the growth or health of the plant. In the alternative, the stabilizing means can contribute to nutritional properties of the liquid composition, such as but not limited to, the levels of nitrogen, phosphorus, or potassium.

In some embodiments, the composition can include between 0.5-1.5% phosphoric acid. In other embodiments, the composition may comprise less than 0.5% phosphoric acid. In some embodiments, the composition can include 0.01-0.3% phosphoric acid. In some embodiments, the composition can include 0.05-0.25% phosphoric acid. In some embodiments, the composition can include 0.01-0.1% phosphoric acid. In some embodiments, the composition can include 0.1-0.2% phosphoric acid. In some embodiments, the composition can include 0.2-0.3% phosphoric acid. In some embodiments, the composition can include less than 0.3% citric acid.

In some embodiments, the composition can include 1.0-2.0% citric acid. In other embodiments, the composition can include 0.01-0.3% citric acid. In some embodiments, the composition can include 0.05-0.25% citric acid. In some embodiments, the composition can include 0.01-0.1% citric acid. In some embodiments, the composition can include 0.1-0.2% citric acid. In some embodiments, the composition can include 0.2-0.3% citric acid.

In some embodiments, the composition can include less than 0.5% potassium sorbate. In some embodiments, the composition can include 0.01-0.5% potassium sorbate. In some embodiments, the composition can include 0.05-0.4% potassium sorbate. In some embodiments, the composition can include 0.01-0.1% potassium sorbate. In some embodiments, the composition can include 0.1-0.2% potassium sorbate. In some embodiments, the composition can include 0.2-0.3% potassium sorbate. In some embodiments, the composition can include 0.3-0.4% potassium sorbate. In some embodiments, the composition can include 0.4-0.5% potassium sorbate.

The present invention involves the use of a microalgae composition. Microalgae compositions, methods of preparing liquid microalgae compositions, and methods of applying the microalgae compositions to plants are disclosed in WO2017/218896A1 (Shinde et al.) entitled Microalgae-Based Composition, and Methods of its Preparation and Application to Plants, which is incorporated herein in full by reference. In one or more embodiments, the microalgae composition may comprise approximately 10%40.5% w/w of Chlorella microalgae cells. In one or more embodiments, the microalgae composition may also comprise one of more stabilizers, such as potassium sorbate, phosphoric acid, ascorbic acid, sodium benzoate, citric acid, or the like, or any combination thereof. For example, in one or more embodiments, the microalgae composition may comprise approximately 0.3% w/w of potassium sorbate or another similar compound to stabilize its pH and may further comprise approximately 0.5-1.5% w/w phosphoric acid or another similar compound to prevent the growth of contaminants. As a further example, in one or more embodiments where it is desired to use an OMRI (Organic Materials Review Institute) certified organic composition, the microalgae composition may comprise 1.0-2.0% w/w citric acid to stabilize its pH, and may not contain potassium sorbate or phosphoric acid. In one or more embodiments, the pH of the microalgae composition may be stabilized to between 3.0-4.0.

In some embodiments and Examples below, the microalgae composition may be referred to as PHYCOTERRA®. The PHYCOTERRA® Chlorella microalgae composition is a microalgae composition comprising Chlorella. The PHYCOTERRA® Chlorella microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at between 65° C.-75° C. for between 90-150 minutes, adding potassium sorbate and phosphoric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration. The PHYCOTERRA® Chlorella microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the PHYCOTERRA® Chlorella microalgae composition may comprise between approximately 0.3% potassium sorbate and between approximately 0.5%-1.5% phosphoric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88.2%-89.2% water. It should be clearly understood, however, that other variations of the PHYCOTERRA® Chlorella microalgae composition, including variations in the microalgae strains, variations in the stabilizers, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be an OMRI certified microalgae composition referred to as TERRENE®. The OMRI certified TERRENE® Chlorella microalgae composition is a microalgae composition comprising Chlorella. The OMRI certified TERRENE® Chlorella microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at between 65° C.-75° C. for between 90-150 minutes, adding citric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration. The OMRI certified TERRENE® Chlorella microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the OMRI certified TERRENE® Chlorella microalgae composition may comprise between approximately 0.5%-2.0% citric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88%-89.5% water. It should be clearly understood, however, that other variations of the OMRI certified TERRENE® Chlorella microalgae composition, including variations in the microalgae strains, variations in the stabilizers, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be an OMRI certified microalgae composition referred to as OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition or as TERRENE65. The OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition is a microalgae composition comprising Chlorella. The OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at 65° C. for between 90-150 minutes, adding citric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration. The OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition may comprise between approximately 0.5%-2.0% citric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88-89.5% water. It should be clearly understood, however, that other variations of the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be an OMRI certified microalgae composition referred to as OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition or as TERRENE90. The OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition is a microalgae composition comprising Chlorella. The OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at 90° C. for between 90-150 minutes, adding citric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration. The OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition may comprise between approximately 0.5%-2.0% citric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88-89.5% water. It should be clearly understood that other variations of the OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be referred to as Aurantiochytrium acetophilum HS399 whole biomass (WB) or HS399 WB. The Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition is a microalgae composition comprising Aurantiochytrium acetophilum HS399. The Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition treatments were prepared by growing the Aurantiochytrium acetophilum HS399 microalgae in non-axenic acetic acid supplied heterotrophic conditions, increasing the concentration of Aurantiochytrium acetophilum HS399 using a centrifuge, pasteurizing the concentrated Aurantiochytrium acetophilum HS399 at between 65° C.-75° C. for between 90-150 minutes, adding approximately 0.3% w/w of potassium sorbate and between approximately 0.5-1.5% phosphoric acid to stabilize the pH of the Aurantiochytrium acetophilum HS399 to between 3.0-4.0, and then adjusting the whole biomass to a desired concentration. It should be clearly understood that other variations of the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be referred to as Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed). The Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition is a microalgae composition comprising Aurantiochytrium acetophilum HS399. The Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition treatments were prepared by growing the Aurantiochytrium acetophilum HS399 microalgae in non-axenic acetic acid supplied heterotrophic conditions, increasing the concentration of Aurantiochytrium acetophilum HS399 using a centrifuge, pasteurizing the concentrated Aurantiochytrium acetophilum HS399 at between 65° C.-75° C. for between 90-150 minutes, adding approximately 0.3% w/w of potassium sorbate and between approximately 0.5%-1.5% phosphoric acid to stabilize the pH of the Aurantiochytrium acetophilum HS399 to between 3.0-4.0, and then adjusting the whole biomass to a desired concentration. Once the Aurantiochytrium acetophilum HS399 microalgae cells were concentrated from the harvest, they were washed; i.e. diluted with water in a ratio of 5:1 and centrifuged again in order to remove dissolved material and small particles. It should be clearly understood that other variations of the Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, variations in the washing method, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be referred to as Aurantiochytrium acetophilum HS399 extracted biomass (EB) or HS399 EB. The Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition is a microalgae composition comprising Aurantiochytrium acetophilum HS399. The Aurantiochytrium acetophilum HS399 extracted biomass (EB) treatments were prepared by growing the Aurantiochytrium acetophilum HS399 microalgae in non-axenic acetic acid supplied heterotrophic conditions, increasing the concentration of Aurantiochytrium acetophilum HS399 using a centrifuge, pasteurizing the concentrated Aurantiochytrium acetophilum HS399 at between 65° C.-75° C. for between 90-150 minutes, adding approximately 0.3% w/w of potassium sorbate and between approximately 0.5%-1.5% phosphoric acid to stabilize the pH of the Aurantiochytrium acetophilum HS399 to between 3.0-4.0, processing the Aurantiochytrium acetophilum HS399 with an oat filler in an expeller process to lyse the cells and separate oil from the residual biomass, and then adjusting the residual biomass to a desired concentration. It should be clearly understood that other variations of the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, variations in the extraction method, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be referred to as a combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition or 25% Chlorella: 75% HS399 WB. The combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition is a microalgae composition comprising Chlorella and Aurantiochytrium acetophilum HS399. For the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition, the Chlorella microalgae cells were cultured in outdoor pond reactors in non-axenic acetic acid supplied mixotrophic conditions and the concentration of Chlorella was increased using a centrifuge. The Aurantiochytrium acetophilum HS399 cells were cultured in non-axenic acetic-acid supplied heterotrophic conditions and the concentration of HS399 was increased using a centrifuge. The concentrated Chlorella cells were then combined with the concentrated HS399 whole biomass cells and adjusted to the desired concentration of 25% Chlorella: 75% HS399 whole biomass (WB). The combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition was then pasteurized at between 65° C.-75° C. for between 90-150 minutes and then stabilized by adding approximately 0.3% w/w of potassium sorbate and between approximately 0.5%-1.5% phosphoric acid to stabilize the pH of the 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition to between 3.0-4.0. It should be clearly understood, however, that other variations of the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the order of the processing steps (blending, pasteurizing, stabilizing), and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be referred to as GP2C. The GP2C Chlorella microalgae composition comprised Chlorella. The GP2C Chlorella microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at between 65° C.-75° C. for between 90-150 minutes, adding potassium sorbate and phosphoric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration. The GP2C Chlorella microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the GP2C microalgae composition may comprise between approximately 0.3% potassium sorbate and between approximately 05%-1.5% phosphoric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88.2%-89% water. It should be clearly understood, however, that other variations of the GP2C Chlorella microalgae composition, including variations in the microalgae strains, variations in the stabilizers, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be referred to as a combination 25% Chlorella: 75% HS399 extracted biomass (EB) microalgae composition, a 50% Chlorella: 50% HS399 extracted biomass (EB) microalgae composition, a 75% Chlorella: 25% HS399 extracted biomass (EB) microalgae composition, or a combination GP2C:399 microalgae composition. The combination GP2C:399 microalgae composition comprises Chlorella and Aurantiochytrium acetophilum HS399 extracted biomass (EB). For the combination GP2C:399 microalgae composition, the Chlorella microalgae cells were cultured in outdoor pond reactors in non-axenic acetic acid supplied mixotrophic conditions and the concentration of Chlorella was increased using a centrifuge; the Aurantiochytrium acetophilum HS399 microalgae cells were cultured in non-axenic acetic acid supplied heterotrophic conditions, the concentration of HS399 was increased using a centrifuge, and the HS399 cells were then processed with an oat filler in an expeller process to lyse the cells and separate oil from the residual biomass. The concentrated GP2C Chlorella whole biomass microalgae cells and the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae cells were blended together to the ratios of 50:50, 25:75, and 75:25, then pasteurized at between 65° C.-75° C. for between 90-150 minutes and then stabilized by adding approximately 0.3% w/w of potassium sorbate and between approximately 0.5%-1.5% phosphoric acid to stabilize the pH of the 25% Chlorella: 75% HS399 extracted biomass (EB) microalgae composition to between 3.0-4.0. It should be clearly understood, however, that other variations of the combination GP2C:399 microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the order of the processing steps (blending, pasteurizing, stabilizing), and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition may be referred to as a Greenwater Polyculture (GWP) treatment. Greenwater Polyculture may be prepared by beginning with a culture of Scenedesmus microalgae that is left outdoors in an open pond and harvested continuously over a year. The culture may comprise anywhere from less than 50% Scenedesmus to greater than 75% Scenedesmus and the concentration varies throughout the year. Other algae may colonize in the GWP as well as other bacteria and microorganisms.

In some embodiments, the composition is a liquid and substantially includes of water. In some embodiments, the composition can include 70-99% water. In some embodiments, the composition can include 85-95% water. In some embodiments, the composition can include 70-75% water. In some embodiments, the composition can include 75-80% water. In some embodiments, the composition can include 80-85% water. In some embodiments, the composition can include 85-90% water. In some embodiments, the composition can include 90-95% water. In some embodiments, the composition can include 95-99% water. The liquid nature and high-water content of the composition facilitates administration of the composition in a variety of manners, such as but not limit to: flowing through an irrigation system, flowing through an above ground drip irrigation system, flowing through a buried drip irrigation system, flowing through a central pivot irrigation system, sprayers, sprinklers, and water cans.

In some embodiments, the liquid composition can be used immediately after formulation, or can be stored in containers for later use. In some embodiments, the composition can be stored out of direct sunlight. In some embodiments, the composition can be refrigerated. In some embodiments, the composition can be stored at 1-10° C. In some embodiments, the composition can be stored at 1-3° C. In some embodiments, the composition can be stored at 3-50° C. In some embodiments, the composition can be stored at 5-8° C. In some embodiments, the composition can be stored at 8-10° C.

In some embodiments, administration of the liquid composition to soil, a seed or plant can be in an amount effective to produce an enhanced characteristic in plants compared to a substantially identical population of untreated seeds or plants. Such enhanced characteristics can include accelerated seed germination, accelerated seedling emergence, improved seedling emergence, improved leaf formation, accelerated leaf formation, improved plant maturation, accelerated plant maturation, increased plant yield, increased plant growth, increased plant quality, increased plant health, increased fruit yield, increased fruit sweetness, increased fruit growth, and increased fruit quality. Non-limiting examples of such enhanced characteristics can include accelerated achievement of the hypocotyl stage, accelerated protrusion of a stem from the soil, accelerated achievement of the cotyledon stage, accelerated leaf formation, increased marketable plant weight, increased marketable plant yield, increased marketable fruit weight, increased production plant weight, increased production fruit weight, increased utilization (indicator of efficiency in the agricultural process based on ratio of marketable fruit to unmarketable fruit), increased chlorophyll content (indicator of plant health), increased plant weight (indicator of plant health), increased root weight (indicator of plant health), increased shoot weight (indicator of plant health), increased plant height, increased thatch height, increased resistance to salt stress, increased plant resistance to heat stress (temperature stress), increased plant resistance to heavy metal stress, increased plant resistance to drought, increased plant resistance to disease, improved color, reduced insect damage, reduced blossom end rot, and reduced sun burn. Such enhanced characteristics can occur individually in a plant, or in combinations of multiple enhanced characteristics.

In some embodiments, a liquid composition can be administered before the seed is planted. In some embodiments, a liquid composition can be administered at the time the seed is planted. In some embodiments, a liquid composition can be applied by dip treatment of the roots. In some embodiments, a liquid composition can be administered to plants that have emerged from the ground. In some embodiments, a liquid or dried composition can be applied to the soil before, during, or after the planting of a seed. In some embodiments a liquid or dried composition can be applied to the soil before or after a plant emerges from the soil.

In some embodiments, the volume or mass of the microalgae based composition applied to a seed, seedling, or plant may not increase or decrease during the growth cycle of the plant (i.e., the amount of the microalgae composition applied to the plant will not change as the plant grows larger). In some embodiments, the volume or mass of the microalgae based composition applied to a seed, seedling, or plant can increase during the growth cycle of the plant (i.e., applied on a mass or volume per plant mass basis to provide more of the microalgae composition as the plant grows larger). In some embodiments, the volume or mass of the microalgae based composition applied to a seed, seedling, or plant can decrease during the growth cycle of the plant (i.e., applied on a mass or volume per plant mass basis to provide more of the microalgae composition as the plant grows larger).

In one non-limiting embodiment, the administration of the composition may comprise contacting the foliage of the plant with an effective amount of the composition. In some embodiments, the liquid composition may be sprayed on the foliage by a hand sprayer, a sprayer on an agriculture implement, or a sprinkler. In some embodiments, the composition can be applied to the soil.

The rate of application of the composition at the desired concentration can be expressed as a volume per area. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 10-50 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the rage of 10-15 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 15-20 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 20-25 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 25-30 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 30-35 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 35-40 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 40-45 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 45-50 gallons/acre.

In some embodiments, the rate of application of the liquid composition in a soil or foliar application can comprise a rate in the range of 0.01-10 gallons/acre. In some embodiments, the rate can be 0.12-4%. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 0.01-0.1 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil or foliar application may comprise a rate in the range of 0.1-1.0 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 0.25-2 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 1-2 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 2-3 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 3-4 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 4-5 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 5-10 gallons/acre.

In some embodiments, the v/v ratio of the composition can be between 0.001%-50%. The v/v ratio can be between 0.01-25%. The v/v ratio of the composition can be between 0.03-10%.

The frequency of the application of the composition can be expressed as the number of applications per period of time (e.g., two applications per month), or by the period of time between applications (e.g., one application every 21 days). In some embodiments, the plant can be contacted by the composition in a foliar application every 3-28 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 4-10 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 18-24 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 3-7 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 7-14 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 14-21 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 21-28 days. In some embodiments, the soil or plant can be treated with the composition once per planting. In some embodiments, the soil or plant can be treated with the composition one time every cutting/harvest.

Foliar application(s) of the composition generally begin after the plant has become established, but can begin before establishment, at defined time period after planting, or at a defined time period after emergence form the soil in some embodiments. In some embodiments, the plant can be first contacted by the composition in a foliar application 5-14 days after the plant emerges from the soil. In some embodiments, the plant can be first contacted by the composition in a foliar application 5-7 days after the plant emerges from the soil. In some embodiments, the plant can be first contacted by the composition in a foliar application 7-10 days after the plant emerges from the soil. In some embodiments, the plant can be first contacted by the composition in a foliar application 10-12 days after the plant emerges from the soil. In some embodiments, the plant can be first contacted by the composition in a foliar application 12-14 days after the plant emerges from the soil.

In another non-limiting embodiment, the administration of the composition can include contacting the soil in the immediate vicinity of the planted seed with an effective amount of the composition. In some embodiments, the liquid composition can be supplied to the soil by injection into a low volume irrigation system, such as but not limited to a drip irrigation system supplying water beneath the soil through perforated conduits or at the soil level by fluid conduits hanging above the ground or protruding from the ground. In some embodiments, the liquid composition can be supplied to the soil by a soil drench method wherein the liquid composition is poured on the soil.

The composition can be diluted to a lower concentration for an effective amount in a soil application by mixing a volume of the composition in a volume of water. The percent solids of microalgae sourced components resulting in the diluted composition can be calculated by the multiplying the original concentration in the composition by the ratio of the volume of the composition to the volume of water. Alternatively, the grams of microalgae sourced components in the diluted composition can be calculated by the multiplying the original grams of microalgae sourced components per 100 mL by the ratio of the volume of the composition to the volume of water.

The rate of application of the composition at the desired concentration can be expressed as a volume per area. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 50-150 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 75-125 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 50-75 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 75-100 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 100-125 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 125-150 gallons/acre.

In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 10-50 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 10-20 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 20-30 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 30-40 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 40-50 gallons/acre.

In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 0.01-10 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 0.01-0.1 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 0.1-1.0 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 1-2 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 2-3 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 3-4 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 4-5 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 5-10 gallons/acre.

In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 2-20 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 3.7-15 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 2-5 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 5-10 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 10-15 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 15-20 liters/acre.

Prior patent applications containing useful background information and technical details are PCT/US2017/053432 titled METHODS OF CULTURING AURANTIOCHYTRIUM USING ACETATE AS AN ORGANIC CARBON SOURCE, filed on Sep. 26, 2017; PCT/US2015/066160, titled MIXOTROPHIC CHLORELLA-BASED COMPOSITION, AND METHODS OF ITS PREPARATION AND APPLICATION TO PLANTS, filed on Dec. 15, 2015; and PCT/US2017/037878 and PCT/2017/037880, both applications titled MICROALGAE-BASED COMPOSITION, AND METHODS OF ITS PREPARATION AND APPLICATION TO PLANTS, both filed on Jun. 16, 2017. Each of these applications is incorporated herein by reference in its entirety.

It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto. All patents and references cited herein are explicitly incorporated by reference in their entirety.

EXAMPLES Example 1

“Degrees Brix” or “Brix” is a metric that is used in the food industry for measuring the approximate amount of sugars in fruits, vegetables, juices, soft drinks, wine, and in the starch and sugar manufacturing industry. Brix usually refers to a scale of measurement for total dissolved solids in the juice of the fruit or vegetable, wherein the dissolved solids are usually sugars and the Brix measurement approximates the sugar content of a sample.

Fruiting plants, as described herein, include any plant that produces a fruit; i.e. the fleshy or dry ripened ovary of the plant which encloses the seed or seeds.

A trial was conducted on strawberry (var. Camarosa) in Winter Garden, Fla. to evaluate performance of the PHYCOTERRA® Chlorella microalgae composition, the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, and the Greenwater Polyculture (GWP) microalgae composition on berry quality after storage, on consumer preference, and sweetness. The trial was transplanted in late October 2016 and harvested through early March 2017. All plots were managed according to the local standard practice (see Study Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Camarosa) Location Winter Garden, FL Transplanting Date Oct. 24, 2016 Pick Frequency Weekly culls when not assessed Bed dimensions 60″ W × 6″ H, 2 rows Planting density 17,424 plants/A Drip irrigation 6″ emitters, 0.3″ applied daily Fertilizer 20-100 lbs 20-20-20 NPK monthly via drip Pesticide Ridomil, Sevin, Dipel and Captec as needed Soil Type Sandy, non-fumigated Plot Size 12 ft sections of 100 ft bed Replication 4 Product applied 0.5 gal/A via drip at planting then every 2 wks

All plots received the standard fertilization regimen used by the grower for these crops excluding biostimulants. The microalgae compositions were added in addition to standard fertilization. Strawberry plants were transplanted to the field. The first product application was at the time of transplanting and then every 14 days afterward until harvest via drip irrigation. The untreated control received the same amount of carrier water as other treatments at the time of each application. Products were shaken well before application and agitated while in the chemigation tank to prevent solids from settling.

Berries from one harvest (day 113) were harvested and either kept in cold storage onsite or shipped cold overnight to a University in New York where they were kept in cold storage. After a period of 4 days in storage, at both sites, the berries were assessed for post-storage quality, particularly sweetness.

Application rates of the PHYCOTERRA® Chlorella microalgae composition, the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, and the Greenwater Polyculture microalgae composition were as detailed in Table 1 below. Raw data is shown in Table 2 below.

TABLE 1 Treatments Application Treatment Rate Number Product gallon/acre T1 Standard Practice (Untreated) N/A T2 PHYCOTERRA ® composition 0.4 T3 PHYCOTERRA ® composition 0.5 T4 PHYCOTERRA ® composition 1 T5 PHYCOTERRA ® composition 2 T6 HS399 Extracted Biomass (EB) 0.4 T7 HS399 Extracted Biomass (EB) 0.5 T8 HS399 Extracted Biomass (EB) 1 T9 HS399 Extracted Biomass (EB) 2 T10 Green Water Polyculture 0.4 T11 Green Water Polyculture 0.5 T12 Green Water Polyculture 1 T13 Green Water Polyculture 2 T14 Seaweed-based Commercial Reference 0.5

TABLE 2 Raw Data % Advantage over % Brix post % Advantage over Rate % Brix post- Standard Comm. shipping/storage Standard Treatment (gal/A) storage 38 F. Practice Ref. 34 F. Practice Comm. Ref. Standard practice 7.13 7.30 Comm. Ref. 0.5 7.97 12% 9.13 25% PhycoTerra 0.4 7.87 10% −1% 8.98 23% −2% PhycoTerra 0.5 8.70 22% 9% 8.83 21% −3% PhycoTerra 1 8.20 15% 3% 8.90 22% −2% PhycoTerra 2 8.33 17% 5% 9.40 29% 3% HS399 EB 0.4 8.05 13% 1% 9.65 32% 6% HS399 EB 0.5 7.70 8% −3% 8.63 18% −5% HS399 EB 1 8.03 13% 1% 9.05 24% −1% HS399 EB 2 7.83 10% −2% 9.38 28% 3% GWP 0.4 7.38 3% −7% 8.65 18% −5% GWP 0.5 7.70 8% −3% 9.40 29% 3% GWP 1 7.55 6% −5% 8.08 11% −12% GWP 2 7.45 4% −6% 8.50 16% −7%

As shown in FIG. 1, relative to standard practice alone (UTC), bi-weekly additions of the PHYCOTERRA® Chlorella microalgae composition at 0.5 gal/A improved berry sweetness (% brix) after shipping and cold storage. For both conditions (38° F. and 34° F. storage), all treatments were sweeter than standard practice (3-32%). PHYCOTERRA® Chlorella microalgae composition at 0.5 gal/A and 2 gal/A showed some of the highest improvements over commercial practice (22-29%). Compared to the seaweed-based commercial reference, PHYCOTERRA® Chlorella microalgae composition at 2 gal/A was 3-5% better for both conditions. The Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, at all rates, was at least 18% sweeter than standard practice after shipping and storage.

Example 2

A trial was conducted on strawberry (var. Seascape) in Fresno, Calif. to evaluate performance of various microalgae compositions on strawberry growth, yield, and post-harvest berry quality, and sweetness; particularly PHYCOTERRA® Chlorella microalgae composition, the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition, the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition, and the combination 25% Chlorella: 75% HS399 extracted biomass (EB) microalgae composition. All plots received the standard local fertilization regimen used by the grower for this crop, excluding biostimulants. The microalgae compositions were added in addition to standard fertilization. Strawberry plants were transplanted to the field in mid-August 2017, according to local commercial practice. The first product application was via drip irrigation at the time of transplanting and then every 14 days afterward through to final harvest. The untreated control received the same amount of carrier water as other treatments at the time of each product application. Products were shaken well before application and agitated, if possible, while in the chemigation tank to prevent solids from settling. Berries were harvested according to local commercial schedule (twice per week during fruiting season). All plots were managed according to the local standard practice (see Study Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Seascape) Location Fresno, CA Row Spacing Conventional Harvest Schedule 4 harvests will be quantified between Sept-Nov. Weekly picks will be performed otherwise and berries discarded to minimize amount of rotten fruit on plants Fumigation Schedule Non-fumigated Plot size minimum 12 ft section after first 2 ft along 76 ft drip line Observations Taken from multiple subsamples per 12 ft section Replication 6 replicate plots for each treatment and untreated control Local Standard Fertility, weed, insect management, etc. Production Standard Management Fungicide application. Record disease Practice management measures

Application rates of the PHYCOTERRA® Chlorella microalgae composition, the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition, the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition, and the combination 25% Chlorella: 75% HS399 extracted biomass (EB) microalgae composition were as detailed in Table 3 below. Raw data is included in the table shown in FIG. 2.

TABLE 3 Treatments Application Treatment Rate Number Product gallon/acre T1a Untreated control (UTC/standard practice) Water T1b Untreated control (UTC/standard practice) Water T2 Seaweed Commercial Reference 0.5 T3 PHYCOTERRA ® 0.25 T4 PHYCOTERRA ® 0.5 T5 HS399 Whole Biomass (WB) 0.25 T6 HS399 Whole Biomass (WB) 0.5 T7 HS399 Extracted Biomass (EB) 0.25 T8 HS399 Extracted Biomass (EB) 0.5 T9 TERRENE ® pasteurized at 65 C. 0.25 T10 TERRENE ® pasteurized at 65 C. 0.5 T11 25% Chlorella: 75% HS399 WB 0.25 T12 25% Chlorella: 75% HS399 WB 0.5 T13 25% Chlorella: 75% HS399 EB 0.25 T14 25% Chlorella: 75% HS399 EB 0.5 T15 Green Water Polyculture 0.25 T16 Green Water Polyculture 0.5

Five, six, eight and nine weeks after transplanting, Seascape strawberries were harvested and assessed for brix at the time of harvest. As shown in FIG. 3-6, for 3 of 4 pick dates, berries from plants receiving the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition (both rates of 0.25 gal/A and 0.5 gal/A) showed increased brix compared to standard practice (2-10%) and the seaweed-based commercial reference (2-6%). Brix for the final harvest (4^(th) pick) was the most affected by the various treatments. Compared to standard practice, the largest increases in brix were observed for 0.25 gal/A of the combination 25% Chlorella: 75% HS399 extracted biomass (EB) microalgae composition (13%), for 0.25 gal/A of the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition alone (12%), for 0.5 gal/A of the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition (10%), and for 0.25 gal/A of Greenwater polyculture (9%).

Example 3

A trial was conducted on strawberry (var. Red Merlin) in Jupiter, Fla. to evaluate performance of the PHYCOTERRA® Chlorella microalgae composition, the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition and GWP on strawberry growth, yield and sweetness. All plots received standard fertilization regimen used by the grower for these crops, excluding biostimulants. Our products were added in addition to standard fertilization. Strawberry plants were transplanted to the field. The first product application occurred at the time of transplanting and then every 14 days afterward until harvest. The first 3 applications were via drench and the remaining were via drip irrigation. The untreated control received the same amount of carrier water as other treatments at the time of each application. The microalgae compositions were shaken well before application and agitated while in the chemigation tank in order to prevent solids from settling. 4 to 6 harvests were completed during the first flush. All plots were managed according to the local standard practice (see Study Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Red Merlin) Location Jupiter, FL Conventional Row 5′ rows with 10″ plant spacing, entire trial Spacing is 12 rows × 270′ Plot size minimum Minimum 5′ × 25′ (5′ bed size) Observations Taken from multiple subsamples per 25′ section Replication 14 treatments × 8 replicates = 112 treatment plots (12 ft sections) Local Standard Fertility, weed, insect management, etc. Production Standard Management Fungicide application. Record disease Practice management measures

Application rates of the PHYCOTERRA® Chlorella microalgae composition, and the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition were as detailed in Table 4 below. Raw data is included in the table shown in Table 5 below.

TABLE 4 Treatments Application Treatment Rate Number Product gallon/acre T1 Untreated control (UTC/standard practice) N/A T2 HS399 Extracted Biomass (EB) 0.3 T3 HS399 Extracted Biomass (EB) 0.5 T4 HS399 Extracted Biomass (EB) 1 T5 HS399 Extracted Biomass (EB) 2 T6 PHYCOTERRA ® 0.3 T7 PHYCOTERRA ® 0.5 T8 PHYCOTERRA ® 1 T9 PHYCOTERRA ® 2 T10 Green Water Polyculture 0.3 T11 Green Water Polyculture 0.5 T12 Green Water Polyculture 1 T13 Green Water Polyculture 2 T14 Seaweed Commercial Reference 0.5

TABLE 5 Treatments % Advantage over Rate % Brix at Standard Comm. Treatment (gal/A) harvest Practice Ref. Standard practice 6.7 Commercial reference 0.5 6.5 −3% PhycoTerra 0.3 6.8 2% 5% PhycoTerra 0.5 6.6 −1% 1% PhycoTerra 1 6.6 −2% 1% PhycoTerra 2 6.7 0% 3% HS399 EB 0.3 6.9 2% 5% HS399 EB 0.5 7.0 4% 7% HS399 EB 1 6.7 0% 3% HS399 EB 2 6.5 −2% 0% Greenwater polyculture 0.3 6.8 2% 5% Greenwater polyculture 0.5 6.9 3% 6% Greenwater polyculture 1 6.5 −3% −1% Greenwater polyculture 2 6.8 2% 5%

As shown in FIG. 7, eight weeks after transplanting, Red Merlin strawberries were harvested and assessed for brix at the time of harvest. Increases in brix were observed but were low overall as the berries were all on the lower end of the spectrum for ripe berries (<7%). Compared to standard practice, the lowest application rate of all treatments (0.3 gal/A) showed a 2% increase. Compared to the seaweed-based commercial reference, advantages for all treatments were greater (3-7%) but the rate response was not as clear.

Example 4

A trial was conducted on strawberry (var. Portola) in Guadalupe Valley, Calif. to evaluate performance of the PHYCOTERRA® Chlorella microalgae composition, the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, the Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition, the OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition, and the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition on strawberry growth, yield, post-harvest berry quality, and sweetness. All plots received standard local fertilization regimen used by the grower for this crop, excluding biostimulants. Our products were added in addition to standard fertilization. Strawberry plants were transplanted to the field in early June 2017, according to local commercial practice. The first product application will be via drip irrigation at the time of transplanting and then every 14 days afterward until harvest. The untreated control received the same amount of carrier water as other treatments at the time of each product application. The microalgae compositions were shaken well before application and agitated, if possible, while in the chemigation tank in order to prevent solids from settling. Berries were harvested according to local commercial schedule. All plots were managed according to the local standard practice (see Study Parameters below). Raw data is shown in Table 6 below.

STUDY PARAMETERS Crop Strawberry (var. Portola) Location Guadalupe Valley, CA Conventional Row 40″ furrow spacing with 24″ wide bed spacing, Spacing and plants on plant lines 12″ apart and plant lines 12″ apart Harvest Schedule As frequently as standard local grower practice with estimated 12-16 picks Fumigation Early May, 32 gal/a PicChlor60 Schedule Plot size minimum 1 double-line bed 45 ft length per plot with 80+ plants per plot Trial Design Randomized Complete Block Observations Taken from 70 plants inside 3 ft buffer zone of each plot end Replication 6 replicate plots for each treatment Local Standard Fertility, weed, insect management, etc. Production Standard Fungicide application. Record disease management Management measures Fungicides will be applied weekly Practice when flowers and fruit are present

TABLE 6 Raw Data Day 5 % Brix % Advantage over Post- % Advantage over Rate at Standard Comm. storage Standard Date Treatment (gal/A) harvest Practice Ref. % Brix Practice Comm. Ref. Sep. 5, 2017 Standard Practice 6.3 7.58 Comm. Ref. 0.50 6.1 −2% 7.88 4% PHYCOTERRA 0.25 6.1 −3% −1% 7.67 1% −3% PHYCOTERRA 0.50 6.2 −2% 1% 8.18 8% 4% Terrene90 0.25 5.9 −6% −4% 7.85 4% 0% Terrene90 0.50 6.2 −1% 1% 7.68 1% −3% HS399 WB 0.25 6.0 −5% −2% 7.45 −2% −5% HS399 WB 0.50 6.2 −1% 1% 7.78 3% −1% HS399 WB 0.25 5.9 −6% −3% 7.98 5% 1% washed HS399 WB 0.50 5.9 −7% −4% 7.67 1% −3% washed Combo 399WB 0.25 6.1 −3% −1% 7.90 4% 0% Combo 399WB 0.50 6.1 −2% 0% 7.85 4% 0% Sep. 16, 2017 Standard Practice 5.7 Comm. Ref. 0.50 5.7 1% PHYCOTERRA 0.25 5.8 3% 1% PHYCOTERRA 0.50 6.0 6% 4% Terrene90 0.25 5.9 4% 3% Terrene90 0.50 5.8 2% 1% HS399 WB 0.25 6.1 7% 6% HS399 WB 0.50 6.0 5% 4% HS399 WB 0.25 5.7 1% −1% washed HS399 WB 0.50 5.8 3% 2% washed Combo 399WB 0.25 6.1 7% 6% Combo 399WB 0.50 5.5 −3% −4% Sep. 25, 2017 Standard Practice 7.2 Comm. Ref. 0.50 7.3 2% PHYCOTERRA 0.25 7.2 1% −2% PHYCOTERRA 0.50 7.3 1% −1% Terrene90 0.25 7.2 0% −2% Terrene90 0.50 7.1 −1% −3% HS399 WB 0.25 7.3 1% −1% HS399 WB 0.50 7.4 3% 0% HS399 WB 0.25 7.1 −1% −3% washed HS399 WB 0.50 7.2 1% −2% washed Combo 399WB 0.25 7.2 0% −2% Combo 399WB 0.50 7.2 0% −2% Sep. 27, 2017 Standard Practice 7.7 Comm. Ref. 0.50 7.7 −1% PHYCOTERRA 0.25 7.7 −1% 0% PHYCOTERRA 0.50 7.7 0% 1% Terrene90 0.25 7.8 1% 2% Terrene90 0.50 7.2 −7% −6% HS399 WB 0.25 7.8 1% 2% HS399 WB 0.50 7.6 −2% −1% HS399 WB 0.25 7.7 −1% 0% washed HS399 WB 0.50 7.6 −2% −2% washed Combo 399WB 0.25 7.8 0% 1% Combo 399WB 0.50 7.8 1% 2% Oct. 6, 2017 Standard Practice 7.6 Comm. Ref. 0.50 7.9 4% PHYCOTERRA 0.25 7.7 1% −3% PHYCOTERRA 0.50 8.2 8% 4% Terrene90 0.25 7.9 4% 0% Terrene90 0.50 7.7 1% −3% HS399 WB 0.25 7.5 −2% −5% HS399 WB 0.50 7.8 3% −1% HS399 WB 0.25 8.0 5% 1% washed HS399 WB 0.50 7.7 1% −3% washed Combo 399WB 0.25 7.9 4% 0% Combo 399WB 0.50 7.9 4% 0% ######## Standard Practice 7.2 Comm. Ref. 0.50 7.7 7% PHYCOTERRA 0.25 8.1 12% 5% PHYCOTERRA 0.50 7.8 8% 1% Terrene90 0.25 8.0 10% 3% Terrene90 0.50 7.5 3% −3% HS399 WB 0.25 7.9 9% 3% HS399 WB 0.50 7.2 −1% −7% HS399 WB 0.25 7.7 6% 0% washed HS399 WB 0.50 7.2 0% −6% washed Combo 399WB 0.25 7.6 6% −1% Combo 399WB 0.50 7.2 −1% −7% Nov. 3, 2017 Standard Practice 6.6 Comm. Ref. 0.50 6.5 −3% PHYCOTERRA 0.25 6.7 1% 4% PHYCOTERRA 0.50 6.7 1% 4% Terrene90 0.25 6.7 1% 4% Terrene90 0.50 6.5 −2% 1% HS399 WB 0.25 6.8 3% 5% HS399 WB 0.50 6.6 −1% 2% HS399 WB 0.25 6.4 −3% −1% washed HS399 WB 0.50 6.5 −3% 0% washed Combo 399WB 0.25 6.4 −3% −1% Combo 399WB 0.50 6.8 2% 5% ######## Standard Practice 7.2 Comm. Ref. 0.50 7.2 −1% PHYCOTERRA 0.25 7.0 −3% −2% PHYCOTERRA 0.50 6.7 −7% −6% Terrene90 0.25 6.6 −9% −9% Terrene90 0.50 7.0 −4% −3% HS399 WB 0.25 6.8 −6% −5% HS399 WB 0.50 7.1 −2% −1% HS399 WB 0.25 7.3 0% 1% washed HS399 WB 0.50 7.1 −3% −2% washed Combo 399WB 0.25 7.4 2% 3% Combo 399WB 0.50 7.2 0% 1%

Portola strawberries were harvested on eight occasions between 12 and 24 weeks after transplanting and assessed for % brix at the time of harvest. Fifteen weeks after transplanting, berries were harvested and stored in cold storage for 5 days and assessed for % brix. Brix at the time of harvest was variable for treatments compared to the control. As shown in FIGS. 8-9, the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition (0.25 gal/A) showed an advantage over standard practice in 4 of 8 harvests (2-7%). The PHYCOTERRA® Chlorella microalgae composition (0.5 gal/A) and the OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition (0.25 gal/A) showed an advantage in only 3 of 8 harvests compared to standard practice (6-8% and 4-10%, respectively). Advantages were more seldom compared to the seaweed-based commercial reference. The Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition (0.25 gal/A) showed an advantage over the seaweed commercial reference in 4 of 8 harvests (2-5%). The PHYCOTERRA® Chlorella microalgae composition (0.5 gal/A) showed an advantage over the commercial reference (4%) in 3 of 8 harvests. For the post-harvest brix, as shown in FIG. 10, the PHYCOTERRA® Chlorella microalgae composition (0.5 gal/A) showed the largest advantage over standard practice (8%). The Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition and the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition also had advantages at both rates (3-5%). Only the PHYCOTERRA® Chlorella microalgae composition (0.5 gal/A) had an advantage over the seaweed-based commercial reference (4%).

Example 5

For the treatments referred to in this Example as Commercial Reference+TERRENE® pasteurized at 65° C., the commercial reference was applied first to the soil at a rate of 20 gal/acre. The TERRENE® pasteurized at 65° C. microalgae composition was then added on top via drip irrigation. The commercial reference was only applied 4 times per season, whereas the TERRENE® pasteurized at 65° C. microalgae composition was applied every 14 days until harvest.

For the treatments referred to in this Example as Commercial Reference+TERRENE® pasteurized at 90° C., the commercial reference was applied first to the soil at a rate of 20 gal/acre. The TERRENE® pasteurized at 90° C. microalgae composition was then added on top via drip irrigation. The commercial reference was only applied 4 times per season, whereas the TERRENE® pasteurized at 90° C. microalgae composition was applied every 14 days until harvest.

A trial was conducted on strawberry (var. Portola-Organic) in Santa Maria, Calif. to evaluate performance of various OMRI certified microalgae compositions on organic strawberry growth, yield, post-harvest berry quality, and sweetness; particularly, the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition, the OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition, the combination OMRI certified TERRENE® pasteurized at 65° C.: microbial-based commercial reference microalgae composition, and the combination OMRI certified TERRENE® pasteurized at 90° C.: microbial-based commercial reference microalgae composition. All plots received standard local fertigation practice, including NEPTUNE'S HARVEST fertilizer and NFORCE fertilizer. A control was added with standard local fertigation practice plus 4 applications of a microbial-based commercial reference product that is standard to this location. Treatments included two versions of an OMRI certified Chlorella microalgae composition that differ by pasteurization temperature (the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition and the OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition), each tested alone and each tested in combination with the microbial-based commercial reference. Strawberry plants (frigo) were transplanted to the field in June 2017, according to local commercial practice. The first product application was via drip irrigation at the time of transplanting and then every 14 days afterward through to final harvest. The untreated control received the same amount of carrier water as other treatments at the time of each product application. The microalgae compositions were shaken well before application and agitated while in the chemigation tank in order to prevent solids from settling. Berries were harvested according to local commercial schedule (twice per week during fruiting season). The timing of the commercial reference applications were once at the time of planting (6/20), once 14-21 days after planting (7/5), once in late July/early August (7/31) and the last in early September (9/11). All plots were managed according to the local standard practice (see Study Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Portola) Location Santa Maria, CA Conventional Row Wide 4-row beds, 64-inches center-to-center; Spacing plants spaced 14 inches apart in each of the four rows Harvest Schedule As frequently as standard local grower practice with estimated 32 picks Fumigation Schedule None (Organic) Plot size minimum 1 four-row bed 25-30 ft length per plot with 80+ plants per plot. Plots will be located away from any field edges with 1-2 commercial buffer beds in between Trial Design Randomized complete block Observations Yield data taken from 40 inside plants, outside 40 combined with inside 40 for post-harvest assessments Replication 6 replicate plots for each treatment and untreated control Local Standard Fertility, weed, insect management, etc. Production Standard Management Standard management practices for Practice organic production. Record disease management measures

Application rates of the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition treatment, the OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition treatment, the combination Commercial Reference+OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition treatment, and the combination Commercial Reference+OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition treatment were as detailed in Table 7 below. Raw data is included in the table shown in Table 8 below.

TABLE 7 Treatments Application Treatment Rate Number Product gallon/acre T1 Standard practice only (UTC) Water T2 Commercial reference (No TERRENE ®) 20 T3 Commercial reference + TERRENE ® 0.5 pasteurized at 65° C. T4 Commercial reference + TERRENE ® 0.5 pasteurized at 90° C. T5 TERRENE ® pasteurized at 65° C. 0.25 T6 TERRENE ® pasteurized at 90° C. 0.25 T7 TERRENE ® pasteurized at 65° C. 0.5 T8 TERRENE ® pasteurized at 90° C. 0.5

TABLE 8 Raw Data % Brix % Advantage over Holding Rate Post- Standard Test Treatment (gal/A) Storage Practice Comm. Ref. 1 Standard 6.56 practice Comm. Ref. 20 6.22 −5% Comm. Ref. + 0.50 6.29 −4% 1% Terrene65 Comm. Ref. + 0.50 6.45 −2% 4% TERRENE90 TERRENE65 0.25 6.45 −2% 4% TERRENE65 0.50 6.24 −5% 0% TERRENE90 0.25 6.25 −5% 0% TERRENE90 0.50 6.47 −1% 4% 2 Standard 8.51 practice Comm. Ref. 20 8.14 −4% Comm. Ref. + 0.50 8.75 3% 7% TERRENE65 Comm. Ref. + 0.50 7.99 −6% −2% TERRENE90 TERRENE65 0.25 8.12 −5% 0% TERRENE65 0.50 8.10 −5% −1% TERRENE90 0.25 8.28 −3% 2% TERRENE90 0.50 8.22 −4% 1% 3 Standard 7.65 practice Comm. Ref. 20 7.81 2% Comm. Ref. + 0.50 7.73 1% −1% TERRENE65 Comm. Ref. + 0.50 7.93 4% 1% TERRENE90 TERRENE65 0.25 7.76 1% −1% TERRENE65 0.50 7.92 4% 1% TERRENE90 0.25 7.89 3% 1% TERRENE90 0.50 7.83 2% 0%

At 11, 14 and 18 weeks after transplanting, berries were harvested and stored in cold storage for 6 days then assessed for % brix after the storage period. Referring to FIG. 11, for the first and second holding test, most treatments had lower brix than standard practice. The exception was the combination Commercial Reference+OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition, which had a 3% advantage. For the final holding test, the combination of Commercial Reference+OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition, the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition (0.5 gal/A) alone, and both rates of the OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgae composition alone had 2-4% advantage over standard practice. By this time, the trial was beginning to be affected by a fungal infection that spread across the entire ranch which may have affected berry quality and given the products more of an advantage. Compared to the commercial reference, advantages were observed for several treatments for the first two holding tests, with the highest being the combination Commercial Reference+OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition (7%).

Example 6

A trial was conducted on strawberry (var. Portola) in Oxnard, Calif. to evaluate performance of various microalgae compositions on strawberry growth, yield, post-harvest berry quality, and sweetness; particularly the PHYCOTERRA® Chlorella microalgae composition, the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition, the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, and the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition. All plots receive standard local fertilization regimen used by the grower for this crop, excluding biostimulants. The microalgae compositions were added in addition to standard fertilization. Strawberry plants were transplanted to the field in July 2017, according to local commercial practice. The first product application was via drip irrigation at the time of transplanting and then every 14 days afterward through to final harvest. The untreated control received the same amount of carrier water as other treatments at the time of each product application. The microalgae compositions were shaken well before application and agitated, if possible, while in the chemigation tank to prevent solids from settling. Berries were harvested according to local commercial schedule (twice per week during the fruiting season). All plots were managed according to the local standard practice (see Study Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Portola) Location Oxnard, CA Conventional Wide 4-row beds, 64-inches center-to-center; Row Spacing plants spaced 14 inches apart in each of the four rows Harvest Schedule As frequently as standard local grower practice with estimated 24 picks Fumigation Local practice (recorded) - timing will be in June Schedule Plot size 1 four-row bed 25 ft length per plot with 80+ minimum plants per plot. Plots will be located away from any field edges with 1-2 commercial buffer beds in between Trial Design Randomized complete block Observations Yield data taken from 40 inside plants, outside 40 combined with inside 40 for post-harvest assessments Replication 6 replicate plots for each treatment and untreated control Local Standard Fertility, weed, insect management, etc Production Standard Standard management practices, including fungicide Management application. Record disease management Practice measures. Fungicides will be applied as necessary (by grower) when flowers and fruit are present

Application rates of the PHYCOTERRA® Chlorella microalgae composition, the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition, and the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition were as detailed in Table 9 below. Raw data is included in the table shown in Table 10 below.

TABLE 9 Treatments Application Treatment Rate Number Product gallon/acre T1 Untreated control (UTC/standard practice) Water T2 Seaweed Commercial Reference 0.5 T3 PHYCOTERRA ® Chlorella composition 0.25 T4 PHYCOTERRA ® Chlorella composition 0.5 T5 HS399 Extracted Biomass (EB) 0.25 T6 HS399 Extracted Biomass (EB) 0.5 T7 HS399 Whole Biomass (WB) 0.25 T8 HS399 Whole Biomass (WB) 0.5 T9 TERRENE ® pasteurized at 65° C. 0.25 T10 TERRENE ® pasteurized at 65° C. 0.5 T11 25% Chlorella: 75% HS399 WB 0.25 T12 25% Chlorella: 75% HS399 WB 0.5

TABLE 10 Raw Data % Advantage over Holding Rate % Brix Post- Standard Comm. Test Date Treatment (gal/A) Storage Practice Ref. 1 Sep. 21, 2017 Standard Practice 7.5 Comm. Ref. 0.50 7.2 −4% PHYCOTERRA ® 0.25 7.3 −3% 1% PHYCOTERRA ® 0.50 7.3 −3% 1% TERRENE ® 65 0.25 6.9 −8% −4% TERRENE ® 65 0.50 7.3 −3% 1% HS399 EB 0.25 7.2 −4% 0% HS399 EB 0.50 7.0 −7% −3% HS399 WB 0.25 7.1 −5% −1% HS399 WB 0.50 7.0 −7% −3% Combo 399WB 0.25 7.2 −4% 0% Combo 399WB 0.50 7.1 −5% −1% 2 ######## Standard Practice 7.2 Comm. Ref. 0.50 7.1 −1% PHYCOTERRA ® 0.25 7.1 −1% 0% PHYCOTERRA ® 0.50 7.6 6% 7% TERRENE ® 65 0.25 7.6 6% 7% TERRENE ® 65 0.50 7.7 7% 8% HS399 EB 0.25 7.3 1% 3% HS399 EB 0.50 7.1 −1% 0% HS399 WB 0.25 7.5 4% 6% HS399 WB 0.50 7.4 3% 4% Combo 399WB 0.25 7.7 7% 8% Combo 399WB 0.50 7.3 1% 3% 3 ######## Standard Practice 8.30 Comm. Ref. 0.50 7.80 −6% PHYCOTERRA ® 0.25 8.30 0% 6% PHYCOTERRA ® 0.50 7.80 −6% 0% TERRENE ® 65 0.25 8.60 4% 10% TERRENE ® 65 0.50 8.40 1% 8% HS399 EB 0.25 8.10 −2% 4% HS399 EB 0.50 8.00 −4% 3% HS399 WB 0.25 8.20 −1% 5% HS399 WB 0.50 8.10 −2% 4% Combo 399WB 0.25 8.50 2% 9% Combo 399WB 0.50 8.30 0% 6% 4 ######## Standard Practice 9.70 Comm. Ref. 0.50 9.80 1% PHYCOTERRA ® 0.25 9.70 0% −1% PHYCOTERRA ® 0.50 9.40 −3% −4% TERRENE ® 65 0.25 10.20 5% 4% TERRENE ® 65 0.50 10.00 3% 2% HS399 EB 0.25 9.90 2% 1% HS399 EB 0.50 9.70 0% −1% HS399 WB 0.25 10.10 4% 3% HS399 WB 0.50 10.40 7% 6% Combo 399WB 0.25 10.30 6% 5% Combo 399WB 0.50 10.40 7% 6% 5 Jan. 18, 2018 Standard Practice 8.10 Comm. Ref. 0.50 8.20 1% PHYCOTERRA ® 0.25 8.50 5% 4% PHYCOTERRA ® 0.50 7.90 −2% −4% TERRENE ® 65 0.25 8.90 10% 9% TERRENE ® 65 0.50 8.40 4% 2% HS399 EB 0.25 8.10 0% −1% HS399 EB 0.50 8.20 1% 0% HS399 WB 0.25 8.10 0% −1% HS399 WB 0.50 8.20 1% 0% Combo 399WB 0.25 8.40 4% 2% Combo 399WB 0.50 8.30 2% 1%

At 11, 15, 19, 21 and 26 weeks after transplanting, berries were harvested and stored in cold storage for 6 to 10 days and then assessed for % brix. As shown in FIG. 12, The OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition had an advantage over standard practice in 4 of 5 holding tests (3-10%). The combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition had an advantage over standard practice in 4 of 5 holding tests (2-7%). Multiple treatments had an advantage over the seaweed commercial reference, but the OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgae composition had the highest (2-10%) advantage in 4 of 5 holding tests.

Example 7

A trial was conducted on bell peppers (var. Capsicum annuum Ace) in a greenhouse in Gilbert, Ariz. to evaluate performance of the PHYCOTERRA® Chlorella microalgae composition on bell pepper sweetness. Capsicum annuum Ace is a bell pepper cultivar grown for both mature green and red ripe fruit in controlled environments. This variety was grown from seed to yield in a horticultural greenhouse and the PHYCOTERRA® Chlorella microalgae composition treatments were administered via manual drench to the soil every 14 days starting at the time of transplanting. The purpose of this experiment was to test whether increasing rates of PHYCOTERRA® Chlorella microalgae composition can establish an optimal application rate to increase percent brix.

Replicates were irrigated with treatments bi-weekly until harvest. Data was collected on fruits within the guidelines of USDA marketable bell pepper standards. Mature green fruit was harvested once a week for 3 weeks. Red ripe fruit was harvested daily for three weeks. Fruits were juiced, and percent brix was recorded using a HI 96801 refractometer. The metric described was percent brix, which is a measure of total dissolved solids in the juice of the fruit and which equates to dissolved sugars and sweetness.

Application rates of the PHYCOTERRA® Chlorella microalgae composition as applied for red ripe and mature green bell peppers were as detailed in Table 11 below. Raw data is included in the table shown in Table 12 below.

TABLE 11 Treatments Replicate Plants Mature Red Treatments: Green Ripe Control 4 4  9 mL/gal 4 4 18 mL/gal 4 4 37 mL/gal 4 4 75 mL/gal 4 4 150 mL/gal  4 4

Replicate plants were given a slow release fertilizer (OSMOTCOTE fertilizer) and irrigated with reverse osmosis (RO) water. Every two weeks replicates were treated with corresponding treatment diluted in city water. At the end of the trial, replicate plant fruits were harvested as either mature green or red ripe. Brix measurements were taken only on qualifying fruit based on USDA standards. Fruits were juiced, and percent brix was taken on individual fruits of each replicate plant. This trial ran for 144 days from seeding to final harvest.

Mature green bell peppers were harvested once a week for three weeks. USDA standard marketable fruits were juiced, percent brix was taken on individual fruits and percent was recorded. Table 12 below shows raw values for percent brix for each treatment and percent change of treatments relative to the control. The 9 ml/gal application rate resulted in a 17% increase in brix of green peppers for the earliest harvest, but no other benefits were observed. FIG. 13 shows results from the first green bell pepper harvest where bell peppers treated with 9 ml/gal of PHYCOTERRA® Chlorella microalgae composition demonstrated a numerical advantage over the control. FIG. 14 shows the results from the second green bell pepper harvest and FIG. 15 shows the results from the third green pepper harvest where no advantage was shown over the control in either harvest.

TABLE 12 Raw Data for Green Bell Pepper Harvest Raw Data % change Raw Data % change Raw Data % change Treatment Harvest 1 Harvest 1 Harvest 2 Harvest 2 Harvest 3 Harvest 3 Control 4.48 5.24 5.20  9 mL/gal 5.26 17.41 5.28 0.76 4.83 −7.05 18 mL/gal 4.36 −2.68 4.98 −4.96 4.83 −7.05 37 mL/gal 4.36 −2.68 5.08 −3.05 4.97 −4.49 75 mL/gal 4.44 −0.89 5.20 −0.76 4.97 −4.49 150 mL/gal  4.18 −6.70 4.70 −10.31 5.17 −0.64

Red ripe bell peppers were harvested daily over three-week intervals. USDA standard marketable fruit were juiced, and percent brix was taken on individual fruit. The average percent brix of all fruits per treatment was taken over three one-week intervals. Table 13 below shows raw values of percent brix and percent change of treatments relative to the control. The majority of the application rates resulted in brix increases in 2 of 3 harvest periods. The 75 mL/gal application increased Brix for red peppers in all 3 harvest periods at 7-14%. FIG. 16 shows results from the first week of harvests, where 9 mL/gal, 75 mL/gal and 150 mL/gal of PHYCOTERRA® Chlorella microalgae composition demonstrated a numerical advantage over the control. FIG. 17 shows results from the second week of harvests, where 18 mL/gal, 37 mL/gal and 75 mL/gal of PHYCOTERRA® Chlorella microalgae composition demonstrated a numerical advantage over control. FIG. 18 shows results from the third week of harvests, where at a level of 0.1, 150 mL/gal of PHYCOTERRA® Chlorella microalgae composition had a statistically significant advantage over the control. 9 mL/gal, 18 mL/gal and 75 mL/gal had a numerical advantage over control.

TABLE 13 Raw Data for Red Ripe Bell Peppers Raw Data % change Raw Data % change Raw Data % change Treatment Harvest 1 Harvest 1 Harvest 2 Harvest 2 Harvest 3 Harvest 3 Control 8.20 8.91 8.07  9 mL/gal 8.82 7.6 8.53 −4.27 9.34 15.74 18 mL/gal 8.24 0.5 9.30 4.33 8.90 10.33 37 mL/gal 8.34 1.7 9.25 3.72 8.02 −0.55 75 mL/gal 9.25 12.8 9.56 7.28 9.25 14.67 150 mL/gal  9.07 10.6 8.88 −0.36 9.58 18.70

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by “about,” where appropriate). All provided ranges of values are intended to include the end points of the ranges, as well as values between the end points.

The description herein of any aspect or embodiment of the invention using terms such as “comprising”, “having,” “including,” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that “consists of”, “consists essentially of”, or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims and/or aspects appended hereto as permitted by applicable law. 

What is claimed is:
 1. A method of increasing sweetness of fruit of a fruiting plant comprising the step of administering to the fruiting plant, seedling, or seed a liquid composition treatment comprising a culture of microalgae, the microalgae comprising at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium cells in an effective amount to increase total dissolved sugars in the fruit of a population of such fruiting plants compared to a substantially identical population of untreated fruiting plants, and wherein the microalgae is pasteurized at a temperature ranging from 50° C. up to 90° C.
 2. The method of claim 1 wherein administering comprises contacting soil in the immediate vicinity of the fruiting plant, seedling, or seed with an effective amount of the liquid composition treatment.
 3. The method of claim 2 wherein the liquid composition is administered at a rate in the range of 0.25-2 gal/acre.
 4. The method of claim 3 wherein the liquid composition comprises between 100 g-800 g per acre of the at least one of the pasteurized Chlorella cells and the pasteurized Aurantiochytrium cells.
 5. The method of claim 2 wherein the liquid composition is administered at a rate in the range of 9 ml-150 ml per gallon.
 6. The method of claim 5 wherein the liquid composition comprises between 0.95 g-15 g per gallon of the pasteurized Chlorella cells only.
 7. The method of claim 1 wherein the liquid composition treatment further comprises phosphoric acid and potassium sorbate.
 8. The method of claim 1 wherein the liquid composition treatment further comprises citric acid.
 9. The method of claim 1 wherein the pasteurized Chlorella cells being administered are pasteurized at a temperature in the range of 65° C.-90° C. and the pasteurized Aurantiochytrium cells being administered are pasteurized at a temperature in the range of 65° C.-75° C.
 10. The method of claim 1 wherein the at least one of the pasteurized Chlorella cells and the pasteurized Aurantiochytrium cells being administered are pasteurized for between 90-150 minutes.
 11. The method of claim 2 wherein the sweetness of the fruit is increased by 2-32% compared to fruit of a substantially identical population of untreated fruiting plants.
 12. The method of claim 1 wherein the pasteurized Aurantiochytrium cells being administered have been subjected to an extraction process to remove oils from the Aurantiochytrium cells.
 13. The method of claim 1 wherein the liquid composition comprises the pasteurized Chlorella cells and the pasteurized Aurantiochytrium cells in a ratio of one of 25:75, 50:50, and 75:25.
 14. The method of claim 1 wherein the pasteurized Aurantiochytrium cells are pasteurized Aurantiochytrium acetophilum HS399 cells.
 15. A method of increasing sweetness of fruit of a fruiting plant comprising the step of administering to the fruiting plant, seedling, or seed a liquid composition treatment comprising a culture of microalgae, the microalgae comprising at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells in an effective amount to increase total dissolved sugars in the fruit of a population of such fruiting plants by compared to fruit of a substantially identical population of untreated fruiting plants, wherein administering comprises contacting soil in the immediate vicinity of the fruiting plant, seedling, or seed with an effective amount of the liquid composition treatment by drip irrigation, and wherein the microalgae is pasteurized at a temperature ranging from 50° C. up to 90° C.
 16. The method of claim 15 wherein the liquid composition comprises the pasteurized Chlorella cells and the Aurantiochytrium acetophilum HS399 cells in a ratio of one of 25:75, 50:50, and 75:25.
 17. The method of claim 15 wherein the pasteurized Aurantiochytrium acetophilum HS399 cells being administered have been subjected to an extraction process to remove oils from the pasteurized Aurantiochytrium acetophilum HS399 cells.
 18. The method of claim 15 wherein the liquid composition comprises between 0.95 g-15 g per gallon of the at least one of the pasteurized Chlorella cells and the pasteurized Aurantiochytrium acetophilum HS399 cells.
 19. A method of increasing sweetness of fruit of a fruiting plant comprising the steps of: providing a liquid composition treatment comprising a culture of microalgae, the microalgae comprising at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium cells; diluting the liquid composition treatment to contain between 0.95 g-15 g per gallon of the at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium cells; and administering the liquid composition treatment to a fruiting plant, seedling, or seed in an effective amount to increase total dissolved sugars in the fruit of a population of such fruiting plants compared to a substantially identical population of untreated fruiting plants, and wherein the microalgae is pasteurized at a temperature ranging from 50° C. up to 90° C.
 20. The method of claim 19, wherein the liquid composition is administered by contacting soil in the immediate vicinity of the fruiting plant, seedling, or seed with an effective amount of the liquid composition treatment. 